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through the copyediting, typesetting, pagination and proofreading process, which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

10.1111/febs.12939

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Discovery and Characterization of Pseudocyclic Cystine-Knot α-Amylase Inhibitors

with High Resistance to Heat and Proteolytic Degradation

Phuong Quoc Thuc Nguyen, Shujing Wang, Akshita Kumar, Li Jian Yap, Thuy Thanh Luu, Julien

Lescar, and James P Tam*

School of Biological Sciences, Nanyang Technological University, Singapore 637551

Correspondence: James P Tam, School of Biological Sciences, Nanyang Technological University,

Singapore; Fax: 65- 6515 1632; Email: jptam@ntu.edu.sg

Running Title: Pseudocyclic Cystine-Knot α-Amylase Inhibitors

Abbreviations

AAI, Amaranth α-amylase inhibitor; TMA, Tenebrio molitor α-amylase; CK, cystine-knot; MALDI-TOF

MS, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry; HPLC, high

performance liquid chromatography; UPLC, ultra performance liquid chromatography; RACE, rapid

amplification of cDNA ends

Keywords: wrightide; pseudocyclics, cystine knot; α-amylase inhibitors, cis proline

Article type : Original Article

Abstract

Obesity and type-2 diabetes are chronic metabolic diseases that could be benefited by the use of α-

amylase inhibitors to manage starch intake. The pseudocyclics, wrightides Wr-AI1 to Wr-AI3,

isolated from an Apocynaceae plant show promising potentials for further development as orally

active α-amylase inhibitors. These linear peptides retain the stability known for cystine knot

peptides in harsh treatment. They are resistant to treatment by heat, endopeptidase or

exopeptidase, characteristics of cyclic cystine knot peptides. Our NMR and crystallography analysis

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also showed that wrightides, currently the smallest proteinaceous α -amylase inhibitors reported,

contain the backbone-twisting cis proline which is preceded by a non-aromatic residue rather than

a conventional aromatic residue. Modeled structure and molecular dynamics study of Wr-AI1 in

complex with yellow meal worm α-amylase suggested that despite similar structure and cystine

knot fold, members of knottin-type α-amylase inhibitors may bind to insect α-amylase via a

different set of interactions. Finally, we showed that the precursors of pseudocyclic cystine knot α-

amylase inhibitors and their biosynthesis in plants follow secretory protein synthesis pathway.

Together, our work provides insights for the use of the pseudocyclic α-amylase inhibitors as useful

leads for developing orally active peptidyl bioactives as well as an alternative scaffold to cyclic

peptides for engineering metabolic-stable human α-amylase inhibitors.

Database

The nucleotide sequences for Wr-AI1 to Wr-AI3 genes have been deposited in the GenBank database

under GenBank accession numbers KF679826, KF679827, KF679828, respectively. The Wr-AI1

solution structure solved of 10 ensembles with the lowest target function is available in Protein Data Bank

(PDB) with accession code 2MAU. The coordinates of Wr-AI1 crystal structure is available in PDB

database with accession code 4BFH.

Introduction

Small disulfide-rich, proteinaceous bioactives are prominently found in toxins, hormones, growth factors,

and protease inhibitors [1]. Many contain a cystine-knot (CK) motif, with a three-disulfide knotted

structure which is formed by two disulfide bonds, together with the connecting backbones, forming an

embedded ring through which the third bond penetrates [2]. Of particular interest in drug development is

the knottin family of CK peptides containing 25 to 45 amino acid residues, and often, possessing protease

inhibitory functions from which the name was derived [3]. Knottins form compact and defined structures

with extensive internal H-bonding, endowing them with resistance to proteolytic degradation by

endopeptidases and denaturation by heat or chemicals as shown by numerous studies including those

using sequencing experiments to determine their primary structures. Certain CK peptides of the knottin

family have further evolved as macrocycles such as cyclotides, harboring cyclic cystine-knot (CCK) with

no termini, a feature that has further bestowed them resistance to degradation by exopeptidases [4].

Cyclotides, generally consisting of 28-37 amino acids, are known be ultra-stable to proteolytic and heat

degradation, and possess robust quality comparable to small-molecule drug candidates. All these features

bode well for developing orally-active peptidyl bioactives.

In a program to identify potentially orally-active peptidyl bioactives to treat metabolic diseases such as

diabetes, we have initiated mass spectroscopy (MS) profiling to identify cysteine-rich peptidyl α-amylase

inhibitors in traditional medicines. Plants and microorganisms produce a diverse group of proteinaceous

α-amylase inhibitors that function in defense pathways. These inhibitors vary greatly in structure and size,

ranging from small peptides (3 kDa), such as Amaranth α-amylase inhibitor (AAI) [5], to large proteins

such as α-AI1, a 23-kDa α-amylase inhibitor from kidney bean (Phaseolus vulgaris) [6]. They are

structurally classified into seven groups: knottin-type, -thionin-like, CM-proteins, Kunitz-type,

thaumatin-like, legume-lectin-like and microbial inhibitors [7]. These classes of α-amylase inhibitors have

attracted attention as tools in agriculture and for anti-diabetic management.

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The smallest proteinaceous α-amylase inhibitor known to date, the 3-kDa AAI, is currently the only

member reported for the knottin-type group. AAI comprises 32 amino acid residues harboring a CK core.

This inhibitor specifically inhibits the yellow mealworm Tenebrio molitor α-amylase (TMA), but is

inactive against human and bovine α-amylases [5]. Although detailed structural study of the inhibition

mechanism of AAI on TMA has been reported, little is known about its knottin-type homologs as well as

their genetic precursors.

Here, we report on the discovery and characterization of a group of linear knottins with characteristics

and potential use in drug development comparable to those of CCK peptides. Using a combination of

proteomic and genomic methods, we identified three AAI-like α-amylase inhibitors, wrightide-amylase-

inhibitors Wr-AI1 to Wr-AI3 from the medicinal plant Wrightia religiosa (Apocynaceae family). We

showed that they are resistant not only to heat treatment and endopeptidase degradation, but also to

exopeptidase. The structure of Wr-AI1 was analyzed in both solution and crystal form by NMR and X-ray

crystallography (to 1.25 Å resolution), respectively. Modeling the Wr-AI1 – TMA complex using

docking and molecular dynamics suggests that α-amylase inhibition by knottins occurs via an overall-

shape-fitting mechanism rather than through a particular set of polar or ionic interaction in the TMA

active site pocket. We also showed that the precursors of knottin-type α-amylase inhibitors contain a

three-domain structure common for CK peptides. Taken together, our study provides new insights into the

sequence, structure and biosynthesis of CK α-amylase inhibitors which could be used as stable scaffold in

engineering human α-amylase inhibitors.

Results

Isolation of α-amylase inhibitors from W. religiosa

Our preliminary MS profiling of crude extract of W. religiosa leaves and flowers revealed strong positive

signals in the mass range of 3–5 kDa, indicative of cysteine-rich peptides (Fig. 1). Thus, we performed

extraction of the putative cysteine-rich peptides from fresh W. religiosa leaves from Vietnam and

Singapore in 50% ethanol and purified them through several rounds of reversed-phase and strong cation-

exchange high-performance liquid chromatography (HPLC). The most abundant peptides from Vietnam

and Singapore leaves were named wrightide-amylase-inhibitors Wr-AI1 and Wr-AI2, respectively. Each

purified wrightide was fully reduced by dithiothreitol and then digested with trypsin and chymotrypsin.

The resulting fragments were sequenced by tandem mass spectrometry and their sequences deduced by

analyzing b- and y-ions (Fig. 2). By genetic analysis, we also obtained the sequence of wrightide Wr-AI3,

which could not be detected in MS profile.

Wrightides Wr-AI1 to Wr-AI3, all 30 amino acid residues in length, contain 6 cysteine, 3 glycine and 2

proline residues. Together, these three residues account for >35% of the sequences. Wrightides share high

sequence homology among each other (93-96%), differing by one or two residues (Fig. 3), and high

sequence homology with AAI (48%).

Solution structure of Wr-AI1 by 1H NMR

Using the distance, dihedral angle, and hydrogen bond restraints derived from 1H NMR experiments, the

solution structures of Wr-AI1 showed that it adopts similar CK scaffold as does AAI with the same three

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disulfide linkages as Cys I-IV, Cys II-V, and Cys III-VI, where Cys III-VI is the penetrating disulfide

bond (Fig. 3A & 3B). The structure contains three short β-strands: Tyr7-Cys8, His19-Cys20, and Gly27-

Ala30, where His19-Ala30 forms a β-hairpin (Fig. 3C). The β-strands are connected by four β-turns, two

pointing towards the N- and C-terminal ends on one side of the molecule, and the other two to the

opposite side. This compact fold is also stabilized by an abundance of intra-molecular hydrogen bonds

(Fig. 3C) as reported in other cysteine-rich peptides such as plant denfensin PhD1 and cyclotide kalata B5

[8, 9]. Moreover, the structure is devoid of N- or C-terminal tails that would extend away from the CK

core stabilized by three disulfide bonds. Around 30% of the amide proton signals remained in the one

dimensional (1D) spectra after 18 h H/D exchange in D2O. These amide protons are identified as

hydrogen bond donors based on the structures. Wr-AI1 contains two prolines, whose conformations were

identified as cis Pro17 and trans Pro23. The proline cis and trans conformations were confirmed by the

observation of NOE crosspeaks HN

Asp16 - H

Pro17 and HN

Gln22 - HPro23, respectively, since the H

strips of

these four residues were not identified from the noise region of H2O around 4.7 ppm.

Structure of Wr-AI1 by X-ray crystallography

Wr-AI1 showed a high propensity to form fiber-like precipitates at neutral pH. Crystals of Wr-AI1

suitable for X-ray crystallography were successfully obtained after 1-day incubation and diffracted to a

1.25 Å resolution at a synchrotron beamline. The complete peptide chain comprising 30 residues was

unambiguously traced (Movie S1), clearly confirming the disulfide connectivity of Cys I-IV, Cys II-V,

and Cys III-VI as determined by NMR experiment. In addition, the crystal structure of Wr-AI1 agreed

with its solution structure ensemble (Fig. 3D) with an average RMSD value of 0.93 Å for backbone

atoms. Minor differences were observed mainly in the loop region and side chain orientations, including

the disulfide bonds. This could be due to the flexibility in solution of the loop region and side chains. This

observation is consistent with the overlapping of the chemical shifts of Hβ

Cys which created ambiguities in

clearly defining the orientation of disulfide bonds by NMR.

A systematic search for homologous structures deposited in the PDB using the DALI server

(http://www.ebi.ac.uk/) returned four homologous structures with a Z factor greater than 3.0 (Fig. 3A).

The structure of Wr-AI1 is most similar to that of AAI (PDB code: 1QFD in its free form and 1CLV as a

complex with the α-amylase from the yellow meal worm): a superposition of 29 α-carbon atoms returns

an RMSD of 1.10 Å with a strict conservation observed for the inhibitor core and disulfide bridges.

Variations between the two structures are confined to the two turns connecting the individual inhibitor

strands that come in contact with the α-amylase upon complex formation. The structure of Wr-AI1 also

resembles those of several spider toxins (Fig. 3F): the Hainan Toxin III and IV (PDB codes 2JTB and

1RYV, respectively), which are neuronal sodium channel inhibitors comprising 33 and 35 amino-acids

[10], and the GXTX-1E high affinity tarantula toxin (PDB code: 2WH9), which is a potassium channel

inhibitor [11].

Further analysis of the crystal structure unambiguously established the two proline residues as cis Pro17

and trans Pro23 (Fig. 4). In the structure of Wr-AI1, the cis peptide bond between Asp16 and Pro17

causes a local backbone twist (Mobius-like structure similar to Mobius cyclotides). This energetically

unfavorable twist is partly stabilized by a strong hydrogen bond between main-chain atoms of Cys15 and

Tyr18 (Table 3 shows the list of intramolecular hydrogen bonds). Previous studies showed that the cis

conformation occurs at higher frequency in X-Pro peptides where X is an aromatic residue [12]. This high

occurrence is explained by the interaction between the aromatic side chain and the proline residue which

gives rise to ring-current-induced shifts for the cis conformers but not the trans conformers in NMR

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experiments. Here we observed a parallel stacking between the phenol ring of Tyr18 and pyrrolidine ring

of Pro17. Significant shifts from the average chemical shifts of Hδ (2.39 with reference average value

3.63) and Hγ (0.71 with reference average value 2.02) were also observed based on the NMR assignments.

The distance between the centers of Pro17 pyrrolidine ring and Tyr18 phenol ring is 4.9 Å. Thus, the

interaction between the aromatic side chain and the pyrrolidone ring could contribute to the stabilization

of Pro17 cis form.

Modeled complex between Wr-AI1 and TMA

The complex between TMA and AAI was previously characterized by Pereira and coworkers using X-

ray crystalloraphy [13]. In this structure, AAI inserts into a V-shaped crevice located at the interface of

TMA domains A and B that forms the active site accommodating the carbohydrate residues. A total of 18

residues of AAI are in contact (distance <4.0 Å) with 24 residues of TMA. Among them, several residues

of the AAI inhibitor occupy or block the entrance to the six carbohydrate-binding subsites at the TMA’s

active site cleft. These residues include Lys4-Arg7, Met12, Tyr27, and Tyr28, all of which are located at

the hydrophilic accessible surface of the inhibitor molecule. Particularly, Arg7 forms a salt bridge with

the catalytic residue Asp287 from TMA. This residue is also involved in a water-mediated hydrogen-

bonding network with two other catalytic residues Glu222 and Asp185 from TMA. To better understand

how Wr-AI1 can inhibit the amylase activity of TMA, we built an atomic model for their interaction using

the AAI-TMA complex as a template (Fig. 5) and assuming an overall conservation of the molecular

orientation of the inhibitors in the TMA active site pocket. With the exception of Gly27 (Wr-AI1

numbering), no residue located at the interface with the enzyme is strictly conserved between Wr-AI1 and

AAI. Upon complex formation the buried surface area is 1831 Å2 which is comparable to the AAI-TMA

complex (2085 Å2) [13]. The network of interactions that stabilizes the Wr-AI1 complex is detailed in

Table 3 and Fig. 6 (Movie S2). Residues Lys4, Glu6, Tyr7 and Thr21 from Wr-AI1 provide hydrogen

bonds with several negatively charged residues from TMA (Fig. 6). This set of H-bonds are preserved in

>95% of all configurations sampled along the last 250 ns of the molecular dynamics simulation. The total

ΔG is -40.56 (1.07) kcal/mol for this comlex. An analysis of the various components contributing to

molecular complex stabilization gives van der Waals: -88.880.93, electrostatics: 138.444.47 and

nonpolar solvation -77.493.79 kcal/mol. This analysis suggests that the enthalpic contribution of the

association between TMA and Wr-AI1 is mainly driven by van der Waals interactions with a smaller

contribution of electrostatic interaction.

Heat and proteolytic stability

To determine whether wrightides would survive degradation in boiling or protease treatment, parameters

important for administering decoctions in traditional medicines, Wr-AI1 was heated at 100C for 1 h or

incubated with chymotrypsin or cacboxypeptidase A for 4 h. More than 95% of Wr-AI1 remaining intact

was observed at the same retention time in the ultra performance liquid chromatography (UPLC) profiles

after heat treatment (Fig. 7A). The MS profiles of corresponding peaks showed that both peaks contained

native Wr-AI1 (m/z 3246) with a small amount of degraded products.

To show that the cystine-knot structure is important for its proteolytic stability, Wr-AI1 was fully reduced

by dithiothreitol and served as the control in chymotrypsin stability assay. A 9-amino-acid linear peptide

was used as the control in cacboxypeptidase A assay. The control peptides were almost completely

hydrolyzed after a 4-hour incubation with chymotrypsin or 1-hour treatment with cacboxypeptidase A at

37C. Under similar conditions, the native peptide Wr-AI1 was resistant to protease degradation with

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>95% of peptides remaining intact (Fig. 7B and 7C). Our results provide strong evidence for the stability

of wrightides against thermal, endopeptidase and exopeptidase treatments.

α-amylase inhibitory activity

We performed inhibition assays with TMA and α-amylases from human saliva, porcine pancreas, and

fungus (Aspergillus oryzae) using the Bernfeld method [14]. Preliminary results showed that both

wrightides Wr-AI1 and Wr-AI2 exhibited inhibitory activities against TMA in a dose-dependent manner

with IC50 of 1.9 and 2.3 μM, respectively (Fig. 7D). Similar to AAI, Wr-AI1 and Wr-AI2 did not inhibit

α-amylases from fungus or mammals at concentrations up to 100 μM.

Biological activity of amylase inhibitors

Cytotoxic, hemolytic, and antibacterial activities of Wr-AI1 and Wr-AI2 were tested. In our experiments,

wrightides did not show appreciable toxic, hemolytic or antibacterial activity at concentrations up to 100

μM.

Cloning of wrightide-encoding genes

Using 3 and 5 RACE (rapid amplification of cDNA ends) PCRs, we obtained the Wr-AI2 full-length

gene from RNA extract from a Singapore plant. Subsequently we used two primers derived from the 5

and 3 untranslated regions of Wr-AI2 clone and successfully amplified DNA sequences of Wr-AI1, Wr-

AI2, and a novel wrightide, Wr-AI3, which was not found at protein level.

Fig. 8 shows the deduced 87-amino-acid precursors of Wr-AI1 to Wr-AI3 and their alignment with

previously characterized CK trypsin inhibitor and -conotoxin precursors. In general, wrightide

precursors contain a 21-residue ER signal sequence followed by a pro-domain of 36 residues and a 30-

residue wrightide domain at the C-terminus. Comparison between RACE and DNA PCRs showed that

wrightide genes contain a phase-one intron in the middle of ER signal. The signal sequence and pro-

domain of wrightide precursors are almost identical except for three residue differences in the pro-domain

and several silent mutations at the gene level as highlighted in Fig. 8A.

Discussion

In this paper, we used proteomic, genomic, and structural methods to characterize the 30-residue knottin-

type α-amylase inhibitors, Wr-AI1 to Wr-AI3, from W. religiosa of the Apocynaceae family. Since the

discovery of AAI in 1994, the 32-residue AAI has remained the only representative of the knottin group

that exhibits α-amylase inhibitory activity [5]. The discovery of wrightides thus extends the list of the

family of knottin α-amylase inhibitors. With two fewer amino acids than AAI, the wrightide family

represents the smallest proteinaceous α-amylase inhibitors reported. Interestingly, these wrightides are

resistant to both heat denaturation and proteolytic degradation, including exopeptidase treatment. Thus,

wrightides take on the favorable stability features of cyclic CK peptides such as cyclotides. In wrightides,

their N- and C-termini are protected by disulfide bonds at the ultimate or penultimate residues. Our

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structural analysis showed that this arrangement enables the termini to loop back to the peptide chain via

disulfide bonds like a “pseudocyclics”, particularly at the N-terminus of wrightides. These pseudocylic

cystine-knot (PCK) peptides, with or without one extra residue flanking at the disulfide-looping terminus,

would likely escape the degradation by exopeptidases

The backbone-twisting cis proline in PCK inhibitors

The presence of cis proline in naturally occurring cysteine-rich peptides generally causes a twist in the

peptide backbone. This was used as a benchmark to classify cyclotides into Möbius (with cis Pro) and the

bracelet (without cis Pro) subfamilies [4]. In this work, we found that PCK wrightide Wr-AI1 also

contains one backbone-twisting cis proline. Also in our unreported work, we found PCK peptides in three

other Apocynaceae plants, each of which containing 3-4 proline residues and four determined by NMR

spectroscopy to have 1-2 cis proline residues. Together, these results suggested that the occurrence of cis

proline bond could be underestimated in proline-rich cysteine-rich peptides.

Surveys of protein databases revealed approximately 35% and 6–8% cis proline in small polypeptides and

native proteins, respectively [15]. The percentage of cis proline amide bond increases to as high as 12–

16% when proline is preceded by an aromatic residue in protein primary sequences. The steric repulsion

of the pyrrolidine rings of a proline residue with the two neighboring Cα atoms generally renders the cis

configuration energetically less favorable than the trans configuration. In peptides with cis proline

preceded by an aromatic residue, clustering of the aromatic side chain and the pyrrolidine ring provides

stability to the sterically constrained cis proline, which is manifested in part by the selective ring-current-

induced shifts of proline Hα and H

β in NMR spectroscopy [12]. Our analysis of the Wr-AI1 structure

demonstrated the occurrence of one cis proline in X-Pro amide bonds, where X is a non-aromatic residue.

The backbone twist caused by this cis proline is likely stabilized by the hydrogen bond between

neighboring residues Cys15 and Tyr18 rather than direct stacking of the preceding aromatic side chain

and proline residue. Thus, the study of local interactions that stabilize the cis Pro residues in Wr-AI1 and

other PCK α-amylase inhibitors may reveal diverse mechanisms of cis Pro formation in cis proline-rich

peptides.

Shape-fitting inhibition mechanism between PCK α-amylase inhibitors and TMA

A model of the interaction between Wr-AI1 and TMA was constructed assuming an overall conservation

of molecular orientation in the TMA’s active site pocket compared to AAI. Interestingly, despite the lack

of sequence conservation between both peptide inhibitors, several side chains that project from the

surface of the two inhibitors are placed in similar positions in the active-site crevice of TMA and small

movements would allow them to form equivalent contacts with the enzyme. Molecular dynamics study of

the modeled complex suggests that Wr-AI1 binds to TMA’s active-site depression via an interaction

network built largely on nonpolar interactions and completely lacking the critical salt bridge observed for

AAI (Table 3). A crystal structure for the TMA-Wr-AI1 complex is needed to confirm this hypothesis.

Wrightides follow the biosynthesis pathway for secretory proteins

Our genetic analysis showed that wrightide precursors consist of an endoplasmic reticulum signal domain

with a phase-one intron, a pro-domain, and a single wrightide domain at its C-terminus. The gene

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organization, starting with a signal peptide, provides hints of the biosynthesis pathway of wrightides

which are gene-encoded and ER-targeted following the conventional pathway for secretory proteins (Fig.

8B) as suggested for many cysteine-rich peptides [16]. The signal peptide is generally removed by SPase

I from the precursor to release the pro-peptide. A single cleavage between the 36-residue pro-domain and

the 30-residue functional domain subsequently produces the native wrightide. These characteristics

distinguish them as ribosomally synthesized peptides from smaller peptides of 5–12 residues that are

synthesized by non-ribosomal multi-enzyme complexes [17, 18].

The three-domain precursor structure is commonly found in many CK peptides, both cyclic and linear.

Examples for such cyclic plant CK peptide precursors include cyclotides from Rubiaceae, Violaceae, and

Solanaceae families [17, 19] and squash trypsin inhibitors from M. cochinchinensis [20] whereas selected

examples for linear plant CK peptide precursors are acyclic cyclotides from Violaceae, Rubiaceae, and

Poaceae families [21-23] and towel gourd trypsin inhibitors [24]. This precursor organization is also used

by animals such as cone snails to encode ion channel blockers ω-conotoxins [25] and δ-conotoxins [26]. It

should be noted that within the plant kingdom, diverse structures are used to organize CK peptide

precursors. One example is precursors with multiple repeats of cyclic or linear CK peptides such as those

reported for cyclotides from Rubiaceae and Violaceae families [17, 19] as well as TIPTOP squash trypsin

inhibitors [20]. Another example is chimeric precursors that encode both CK peptides and other types of

proteins, such as cliotide precursors encoding both cyclotides and legume albumin PA1a in C. ternatea

plant [27, 28]. Given such diversity of precursor organization even within a CK peptide family,

understanding about the genetic sequences of each CK peptide family, here PCK α-amylase inhibitors, is

thus beneficial for their applications in crop protection and also provides insights into their biosynthesis.

Knottin-type α-amylase inhibitors with applications in engineering peptidyl bioactives

The CK structure has been employed in nature as scaffolds of a variety of unrelated protein families

found in microbes, animals, and plants. Particularly, α-amylase inhibitors adopting CK fold are small,

extraordinarily stable against heat, endopeptidase as well as exopeptidase degradation, and highly tolerant

to sequence variation [29]. Thus, small CK peptides such as wrightides with MW 3-5 kDa contain

appealing features as potential peptide therapeutics [30, 31]. First, the small size renders wrightides more

accessible to chemical synthesis [32, 33]. Second, the CK peptides in general are highly tolerant to

sequence variations and the spacing of the half cystines, allowing α-amylase inhibitors to potentially serve

as a scaffold for protein engineering to attain new functions, such as in the successful grafting of the

bradykinin-antagonist peptides DALK or DAK onto cyclotide kalata B1 scaffold [34].

A potential application of considerable interest to drug development is the engineering of wrightides to be

orally active mammalian α-amylase inhibitors in treating obesity and type-2 diabetes mellitus (T2DM).

Literature precedents showed that extended hydrophobic interactions could be important for AAI

inhibition on mammalian α-amylases [35]. Human salivary α-amylase (HSA, PDB code: 3DHP) and

TMA share high sequence homology (65%) and structural homology (Z-score of 57.1 with 468 equivalent

residues at rmsd of 1 Å, by DALI server). Superimposing HSA:Wr-AI1 complex with TMA:Wr-AI1

complex reveals four additional loops present in HSA at the interface of the active site, including loops

Asn53-Phe55, Asn137-Gly146, Gly304-Ala310, and Trp344-Val358. The conformational flexibility of

these loops might be responsible for the low affinity binding of Wr-AI1 to HSA. Our docking

experiments suggested that it is possible that careful incorporation of aromatic and positively charged

residues into wrightide templates could improve their contact with the negatively charged enzyme active

sites to render wrightides active against mammalian α-amylases. In this regard, our work showing the

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interaction of Wr-AI1 and TMA provides new insights for a structure-guided approach to design

potentially useful orally active α-amylase inhibitors for managing T2DM and obesity.

Experimental procedures

Isolation of α-amylase inhibitors

800 g of W. religiosa leaves was homogenized and extracted twice in 50% (v/v) ethanol. After

centrifugation (7000 rpm, 10 min), the supernatant was partitioned with dichloromethane. The aqueous

upper layer was concentrated, filtered, and loaded onto a C18 flash column (Grace Davison, US). Elution

was done with increasing concentrations of ethanol. The presence of cysteine-rich peptides in all fractions

was monitored by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-

TOF MS). To purify individual peptides, several dimensions of strong cation exchange and reversed-

phase HPLC were employed.

De novo sequencing with MALDI-TOF MS/MS

About 40 μg of each purified peptide was dissolved in 50 mM ammonium bicarbonate buffer (pH 7.8)

containing 50 mM dithiothreitol and incubated at 37C for 2 h. Digestion with endoproteinase Glu-C,

trypsin or chymotrypsin was carried out at room temperature for 5 min and subsequently sequenced by

MALDI-TOF MS/MS as previously described [27]. Isobaric residues were assigned based on gene

sequences for Wr-AI1 and Wr-AI2 and confirmed based on X-ray or NMR structure for Wr-AI1. The

peptide Wr-AI3 was only determined at gene level.

Solution structure determination using NMR spectroscopy

The NMR sample was prepared by dissolving lyophilized Wr-AI1 into 95% H2O/5% D2O or 99.9% D2O

directly (~1 mM protein and pH/pD 3.3). All NMR experiments were carried out on a Bruker 600 MHz

NMR spectrometer equipped with a cryogenic probe. Two dimensional (2D) total correlation

spectroscopy (TOCSY) and nuclear Overhauser spectroscopy (NOESY) experiments were performed

with mixing times 80 ms and 200 ms, respectively [36]. The 2D data were acquired at 298 K. Water

suppression was achieved using modified WATERGATE pulse sequences [37]. The NMR spectra were

processed with NMRPipe software [38]. The amides involved in hydrogen bonding were identified by the

hydrogen-deuterium exchange 1D 1H experiment [39].

Sequence specific assignments were achieved based on the 2D TOCSY and NOESY, and NOEs were

assigned from the 2D NOESY using the software NMRspy

(http://yangdw.science.nus.edu.sg/Software&Scripts/NMRspy/index.htm). The chemical shifts are

deposited in BioMagResBank (accession number: 18983). Distance restraints were derived from the peak

intensities of the assigned NOEs. Dihedral angles φ were obtained from 3JHN-Hα coupling constants

measured from the 1D 1H spectrum. Hydrogen bond restraints were incorporated based on the observation

of amide protons in the 1D 1H spectra recorded after resuspending the lyophilized Wr-AI1 in D2O for up

to 18 h at 25C.

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Structure was calculated with simulated annealing approach with CYANA 2.0 [40]. Distance restraints

are divided into three classes: 1.8<d<3.4 Å (strong NOEs), 1.8<d<4.2 Å (medium NOEs) and 1.8<d<5.5

Å (weak NOEs). Disulfide bond restraints of 2.0<d (Sγi, S

γj)<2.1 Å, 3.0<d (C

βi, S

γj)<3.1 Å and 3.0<d (S

γi,

Cβ

j)<3.1 Å were employed for structure calculation. During the structure calculation, hydrogen bond

restraints of 1.8-2.2 Å for the NH-O distance and 2.2-3.2 Å for the N-O distance were applied on nine

identified hydrogen bonds according to the slowly exchanging amide protons. Φ angles were constrained

to the range of -150° to -90° for 3JHN-Hα>8 Hz. Structures were displayed and analyzed using software

PyMOL and program PROCHECK-NMR, respectively [41]. The experimental and structural statistics are

summarized in Table 1.

Crystal structure determination

Using sitting drop vapor diffusion method, the native crystals were obtained from the mixture of 1 µl of

Wr-AI1 solution (4.8 mg/ml) and 1 µl of precipitant solution (3.6 M sodium formate, 10% glycerol) after

one-day incubation at 16C. The crystals were stabilized in the precipitant solution supplemented with

40% (v/v) glycerol and flash-frozen in liquid nitrogen. Diffraction intensities to 1.25 Å resolution were

collected at 100 K at the Swiss Light Source Beamline PXIII using a Dectris Pilatus 6M detector.

Integration, scaling and merging of intensities were carried out using programs XDS [42] and SCALA

[43] from the CCP4 suite [44]. Data collection statistics are summarized in Table 2.

The structure was determined by molecular replacement using the program PHASER [45]. The search

probe was the structure of AAI (PDB code 1CLV [13]). The program Arp-Warp [46] was used for chain

tracing and map improvement and the resulting model was corrected manually (Table 2). The

Ramachandran plot calculated using PROCHECK [47] revealed that 87% of the residues were in the most

favoured region and 13% of the residues in additional allowed region.

Molecular docking and molecular dynamics study of the Wr-AI1-TMA complex

To investigate the stability of the TMA:Wr-AI1 complex obtained from docking (which was initially

obtained by simply superimposing Wr-AI1 onto AAI in the TMA-AAI crystal structure), we performed

three molecular dynamics simulation of 500 ns each, using ACEMD [48] and an all atom ff12SB force

field parameters. Hydrogen atoms were added to this initial complex using the Xleap module of AMBER

[49]. The system was solvated with TIP3P water molecules to form a box with at least 10 Å separating

the solute atoms and the edge of the box. A total of 92 Na+ and 70 Cl

- ions, corresponding to a salt

concentration of 150 mM were added to the system by replacing water molecules in random position.

Before the dynamic simulations, the solvated system was relieved of any unfavourable interactions by

subjecting it to 100 steps of energy minimization. Harmonic restraints during the equilibration were

placed on C 1 kcal.mol-1

.Å2 to the energy minimized co-ordinates. The system was heated

up to 300 K in steps of 100 K, followed by the gradual removal of the positional restraints and a 10 ns

unrestrained equilibration at 300 K. Analysis of the resulting trajectories revealed that the simulated

complex reached stability after 50 ns, with RMSD less than 1.8 Å. The first 10 ns of simulation were

performed in NPT, the production run of 500 ns was performed in NVT. The simulation temperature of

300 K was set using Langevin dynamics, with a collision frequency of 0.1 ps-1

. The pressure was

maintained at 1 atm using weak coupling with a pressure relaxation time of 1 ps. During the simulation,

all long range electrostatic interactions were treated with particle mesh Ewald methods [50] using a real

space cut-off distance of 9 Å. Bonds involving hydrogen atoms were constrained using the M-SHAKE

algorithm [51]. A time step of 4 fs with hydrogen mass repartitioning was used and coordinates were

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saved every 100 ps. Hydrogen bond analysis was performed using the PTRAJ module in AMBER for the

last 250 ns of the stabilised trajectory using a distance cut off of 3.5 Å. Binding energy analysis based on

MM/GBSA [52] was performed on the simulated trajectory to calculate the free energy of binding of Wr-

AI1 peptide to TMA. For binding energy calculation, a total of 100 structures were extracted at regular

intervals from the last 250 ns of the trajectory. A salt concentration of 150 mM and Born implicit solvent

model of 2 (igb=2) [53] was used. The binding surface area was calculated using NACCESS software

[54]. Simulation trajectories were visualized in VMD [55] and figures were generated using PyMol.

Heat stability test

Purified Wr-AI1 was heated in boiling water for 1 h and then subjected to UPLC. The peptide Wr-AI1

without heat treatment was used as a control. Collected peaks from UPLC were monitored by MALDI-

TOF MS.

Proteolytic stability test

Purified Wr-AI1 was incubated with or without chymotrypsin (at a final peptide:enzyme ratio of 10:1

mol/mol) in 20 mM ammonium bicarbonate (pH = 7.8) at 37C for 4 h. Purified Wr-AI1 completely

reduced with 50 mM dithiothreitol (2 h, 37C) was treated in the same way and used as a control. Treated

samples or controls were subjected to UPLC and the collected peaks were monitored by MALDI-TOF

MS.

In cacboxypeptidase A stability assay, Wr-AI1 was incubated with or without enzyme (at final

peptide:enzyme ratio of 40:1 mol/mol) in 50 mM Tris-HCl, 1 M NaCl (pH 7.5) at room temperature for

up to 24 h. A linear 9-residue peptide was used as a control. Degradation products were monitored by

UPLC and MALDI-TOF MS.

Assay for α-amylase activity

Alpha-amylase was isolated from yellow mealworm, larvae of Tenebrio molitor, following the procedure

described previously [56]. Assays for α-amylase were carried out in 96-well plates following Bernfeld

method [14]. Enzyme TMA with or without treatment with peptides (20 min, 37C) was incubated with

1% starch (in 20 mM sodium phosphate buffer, pH 6.7) for 5 min. Color reagent (3,5-dinitrosalicilic acid

and sodium potassium tartrate, Sigma) [57] was dispensed into each well and color developed for 20 min

at 100C. Absorbance at 540 nm was read to determine the α-amylase activity. Similar inhibition

experiments were performed for human salivary, porcine pancreatic and Aspergillus oryzae α-amylases

(Sigma).

Hemolysis assay

Fresh type AB blood was donated by a healthy volunteer. The hemolysis assay was performed as

described elsewhere [27].

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Cytotoxicity assay

Cytotoxicity of the purified wrightides was tested using PrestoBlue™ Cell Viability Reagent (Invitrogen).

African green monkey kidney (Vero) cells seeded onto 96-well plate were incubated with Wr-AI1 and

Wr-AI2 at 1 to 100 μM for 24 h at 37C. After incubation, the wells were dispensed with Presto Blue

reagent and left at 37C for 2 h. The fluorescence was subsequently read as instructed by the

manufacturer. 1% Triton X-100 solution was used as positive control.

Antibacterial assay

The antibacterial activity of wrightides was assessed using radial diffusion assay as described previously

[58] on Gram negative Escherichia coli (FDA strain Seattle 1946) and Gram positive Staphylococcus

aureus bacteria. D4R, an in-house peptide dendrimer with potent antibacterial activity, was used as a

positive control. The experiments were done in duplicate.

Cloning of α-amylase inhibitor genes

Total RNA extraction was performed using PureLinkTM

Mini RNA purification kit (Invitrogen) with

additional 3% of 2-mercaptoethanol and 4% of polyvinylpyrrolidone added to the lysis buffer. Total RNA

extract of Singapore W. religiosa leaves was subsequently converted to 3' and 5' RACE cDNA libraries

using 3' RACE System for Rapid Amplification of cDNA Ends (Invitrogen) and SMARTer™ RACE

cDNA Amplification Kit (Clontech), respectively. 3' RACE PCR products using the degenerate primer

targeting the sequence CAQKGE (5'-TGTGCTCArAArGGnGA-3') were gel-purified, cloned into

pGEM®-T Easy Vector (Promega) and sequenced. A reverse primer based on the newly obtained partial

sequence was designed to reveal the remaining encoding gene in 5' RACE PCR. To determine the DNA

sequences of wrightide genes, we performed PCR on W. religiosa DNA extract using two primers:

Wr2speF (5'-TAGGCGCAAACAACATGGCT-AAGC-3') and Wr2speR (5'-CCACATAGCTCG-

TAGAACAAGCTTACAG-3'). The endoplasmic reticulum signal peptides were predicted using SignalP

3.0 (http://www.cbs.dtu.dk/services/-SignalP-3.0/).

Acknowledgments

We thank Giang K.T. Nguyen, Teo C. H. and Lam Y. S. for technical assistance in this project. This

research was supported in part by the Competitive Research Grant by National Research Foundation in

Singapore (NRF-CRP8-2011-05)

Author contribution

PQTN performed or was involved in all the experiments presented in this paper except NMR experiment;

SW performed NMR spectroscopy experiment and sequence calculation; AK analyzed MD results and

enzyme alignment; LJY performed X-ray crystallization experiment; TTL contributed to peptide and

enzyme extraction; JL analyzed X-ray crystallography data, built the structure and modeled the complex;

JPT analyzed the data. PQTN, JL, and JPT contributed mainly to manuscript preparation. All the authors

discussed the results and contributed to the writing of the manuscript.

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References

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Supporting information

Movie S1. Stick representation of Wr-AI1 with 2Fo-Fc map overlaid. The complete chain was

unambiguously traced and contains three disulfide bonds in a cystine-knot motif.

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Movie S2. Movie of MD simulation trajectory of TMA bound to Wr-AI1. The TMA protein (white) and

Wr-AI1 (blue) are shown as "cartoon". Interacting residues from TMA (Asp225, His189, Asp185,

Val151, and Asp287) and Wr-AI1 (Lys4, Glu6, Tyr7, Tyr11 and Thr21) are shown as sticks. See also

Table 3. For clarity purpose, hydrogen atoms are not shown.

Table 1. NMR experimental and structural statistics of Wr-AI1

NOE constraints 681

Intra-residue(|i-j|=0) 332

Sequential (|i-j|=1) 193

Medium-range (1<|i-j|<5) 36

Long-range (|i-j|≥5) 120

Dihedral angle restraints 10

Hydrogen bonds 7

PROCHECK-NMR Ramachandran plot (%)

Most favored region 70.9

Additionally allowed region 28.7

Generously allowed region 0.4

Disallowed region 0

Average maximum violations per structure

Distance (Å) 0.02±0.002

Van der waals (Å) 3.7±0.4

Torsion angles (°) 0.25±0.11

CYANA target function value (Å2) 1.35±0.15

Average RMSD to mean structure (Å)

All back bone atoms (1-30) 0.38±0.08

All heavy atoms (1-30) 0.85±0.12

Table 2. Crystallography data collection and refinement statistics of Wr-AI1

Statistics of data collection

Space group

Cell parametersa P212121

a,b,c (Å) a=16.19, b=29.13, c=47.70

α,β,γ (o) α =β=γ=90

Resolution range (Å) 29.1 – 1.25 (1.28-1.25)

Observed reflections 21,115 (2,864)

Unique reflections 6,362 (856)

Completeness (%) 95.4 (90.3)

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Multiplicity 3.3 (3.3)

Rmergeb 0.029 (0.071)

Mean I/σ(I) 23.2 (11.9)

Statistics of refinement

Resolution range (Å) 24.88 – 1.25 (1.28-1.25)

Number of reflections used for refinement 6,017 (410)

Number of reflections for Rfree calculation (5%) 317 (21)

Rfactor (%)#, Rfree (%)

* 15.8, 15.9

Number of non hydrogen atoms 223

Water molecules 41

Mean B-factor (Å2)

Protein whole chain 5.4

Water 32.9

r.m.s. deviations from ideality

Bond lengths (Å) 0.020

Bond angles (°) 1.85

Ramachandran plot (%)

Most favoured regions 87

Additional allowed regions 13

G-factor$ 0.07

aThe numbers in parentheses refer to the last (highest) resolution shell.

bRmerge = ΣhΣi|Ihi-<Ih>|/Σh,i Ihi, where Ihi is the i

th observation of the reflection h, while <Ih> is its mean

intensity. #Rfactor = Σh||Fobs(h)| - |Fcalc(h)|| / Σ |Fobs(h)|

*Rfree was calculated with 5% of reflections excluded from the whole refinement procedure.

$G factor is the overall measure of structure quality from PROCHECK.

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Table 3. Potential intramolecular hydrogen bonds in Wr-AI1 and intermolecular interactions between

pseudocyclic cystine knot inhibitors and TMA (distance <3.4 Å). Intramolecular distances were

determined by X-ray crystallography. Intermolecular distances between AAI and TMA were derived from

the crystal complex of AAI and TMA (PDB 1CLV) whereas the distances between Wr-AI1 and TMA

were calculated from the molecular dynamics simuation of TMA:Wr-AI1 complex for the last 250 ns of

the trajectory.

Wr-AI1 Distance

(Å)

Wr-AI1 Distance

(Å) Residue/Atom Residue/Atom Residue/Atom Residue/Atom

A2 N Q13 O 2.91 S9 OG L12 N 3.06

A2 O C15 N 2.84 V10 N P23 O 2.97

Q3 N E6 OE1 2.87 C15 O Y18 N 2.80

Q3 O G5 N 3.30 C15 O P17 N 3.31

Q3 O E6 N 3.00 P17 O H19 NE2 3.21

K4 NZ A30 OXT 2.88 H19 N A30 O 2.91

G5 N C29 O 2.76 H19 O A30 N 3.01

E6 O C29 N 2.93 C20 O Q22 N 3.35

C8 N G27 O 2.99 T21 N I28 O 2.92

C8 O G27 N 3.01 T21 OG1 I28 N 3.01

S9 N L12 O 3.02 Q22 O G26 N 2.79

S9 O Y11 N 3.15 Q22 O G27 N 3.00

S9 O L12 N 3.32 Q22 OE1 V24 N 2.94

S9 O L12 O 3.03 Q22 OE1 I25 N 3.01

S9 OG Y11 N 3.06

AAI Distance

(Å)

TMA Distance

(Å)

Wr-AI1 Occupancy

(%) Residue/atom Residue/Atom Residue/Atom

C1 N 2.80 N137 O

K4 NZ 2.73 Q295 OE1

K4 NZ 3.27 Q295 NE1

N6 OD1 2.81 K188 NZ

D13 O 2.93 E135 OE1

T24 O 3.18 N331 ND2

D332 OD2

S25 OG 2.76 D332 OD1

N30 ND2 3.19 D287 O

N30 ND2 3.06 R290 O

S32 OG 3.09 G292 N

V151 O 2.75 Y11 OH 49.89

D185 OD2 2.80 Y7 OH 99.09

H189 NE2 2.75 E6 OE1 97.04

D225 O 2.81 K4 NZ 99.07

D225 OD1 2,81 K4 NZ 48.70

D287 OD2 2.64 T21 OG1 98.44

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Fig. 1. Tissue- and region-specific expression profiles of wrightides from flower and leaf of W. religiosa

plant. 50% ethanol extract of 1 g of each plant sample was purified using C18 solid phase extraction

column. The eluate with 80% acetonitrile was profiled using MALDI-TOF MS to determine the

occurrence of putative CK peptides in different W. religiosa plant parts from Singapore and Vietnam.

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Fig. 2. Tandem MALDI-TOF/TOF MS/MS profiles of two tryptic fragments (m/z 2072 - A and 1198 -

B), provide the full wrightide Wr-AI1 sequence. Ile/Leu assignment was determined by genetic sequence

as well as X-ray and NMR structures.

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Fig. 3. The 3D structure of Wr-AI1. A, Secondary structure illustration against the sequence of Wr-AI1.

The amino-acid sequence of Wr-AI1 is aligned with sequences of: AAI, HT-III(Hainan toxin-III), HT-

IV(Hainan toxin-IV) and GxTX-1E (Guangxiensis toxin-1E) with PDB codes of 1QFD, 2JTB, 1RYV

and 2WH9, respectively. β stands for beta-strand; yellow bridge indicates disulfide bonds and red turn

depicts beta hairpin. B, Solution structure of Wr-AI1 (PDB code: 2MAU). C, Illustration of

intramolecular hydrogen bonds. D, Backbone trace alignment of the crystal structure of Wr-AI1 (blue,

PDB code: 4BFH) with the ten solution structure ensemble (tan). E, Superposition of the solution

structures of Wr-AI1 and AAI. F, Structure alignment of Wr-AI1 with its structural homologs. The figure

was prepared with the program PyMOL.

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Fig. 4. Ribbon diagram and structural features of Wr-AI1. A, Ribbon presentation of Wr-AI1 crystal

structure. Pro17 and Pro23 adopt the cis and trans conformations, respectively. B, Stereoview of the

electron density (2Fobs-Fcalc) of the backbone twist caused by cis Pro17. The electron density was

contoured at 1.0 σ. C, Schematic hydrogen bonding network of the three beta strands of Wr-AI1. The

residues involved in the three beta strands are shown as blue spheres and relevant residues as grey

spheres. The figure was prepared with the program PyMOL.

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Fig. 5. Cartoon and surface view of superimposition of AAI and Wr-AI1 in complex with TMA. A, The

complex model of AAI (magenta) and Wr-AI1 (cyan) with TMA (gray) derived from the AAI-TMA

complex (PDB 1CLV). B and C, Zoom-view of the binding region between AAI (B)/Wr-AI1 (C) and

TMA. Residues of AAI/ Wr-AI1 with atoms within 6 Å away from TMA are colored in magenta/cyan,

while residues of TMA with atoms within 6 Å away from AAI / Wr-AI1 are colored in blue/red,

respectively. Residues that are >6 Å away from TMA in both AAI/ Wr-AI1 are colored orange. D, The

superimposition of AAI and Wr-AI1 at the active site of TMA. Three disulfides between CysI-IV, CysII-

V, and CysIII-VI arranged in a cystine knot motif are highlighted in yellow. E and F, Close-ups of AAI

(E) and Wr-AI1 (F) residues at TMA’s active site. The figure was prepared with the program PyMOL.

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Fig. 6. Stable hydrogen bond interactions between Wr-AI1 (residues displayed as sticks in cyan) and

TMA (residues shown as green sticks) following molecular dynamics simulation (see text)

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Fig. 7. Stabilities and α-amylase inhibitory activity of wrightides. A, Thermal stability of Wr-AI1 and

Wr-AI2. The minor peak after heat treatment contained mainly Wr-AI1/Wr-AI2 with a small amount of

degraded products as examined by MS profile. B, Chymotrypsin stability of wrightide Wr-AI1. C,

Cacboxypeptidase A stability of Wr-AI1. D, Inhibition of wrightides against Tenebrio molitor α-amylase.

Peptides were pre-incubated with TMA for 20 min at 37C. Hydrolysis was started by adding 1% starch.

The reaction was allowed for 5 min, and stopped by adding a color reagent containing DNS. The IC50

values are 1.9 μM and 2.3 μM for Wr-AI1 and Wr-AI2, respectively. The error bars show SDs.

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Fig. 8. Precursor structure of wrightides Wr-AI. A, Alignment of wrightide precursors with precursors of

other CK peptides including -conotoxin SO-3 and towel gourd trypsin inhibitor TGTI-II. The

endoplasmic reticulum signal peptide was assigned using SignalP 3.0 for -conotoxin SO-3 while this

domain is not recognized by SignalP 3.0 for TGTI-II. B, Secretory protein synthesis pathway for

wrightides.